Dentinal tubules of 27 cylindrical bovine root specimens were infected with Enterococcus faecalis. In nine specimens, 5% chlorhexidine was placed in a slow-release device (Activ Point) for 7 days, in another nine irrigation with 10 ml of 0.2% chlorhexidine was used, and the remaining nine served as positive control. Powder dentin samples obtained from within the canal lumina using ISO 025 to 033 burs were examined for the presence of vital bacteria by inoculating brain-heart infusion plates and counting colony forming units. Results were analyzed using analysis of variance and covariance with repeated measures. Heavy bacterial infection was observed at the layer close to the lumen in the control specimens, decreasing rapidly from layer to layer up to the deepest layer tested (400-500 microm), which contained several hundred colony forming units. Viable bacteria in each layer of dentin were significantly reduced with chlorhexidine irrigation solution (p < 0.01) and were completely eliminated with the chlorhexidine slow-release device (p < 0.01).